Nitric Oxide (NO) is a gas that has multiple signalling and effector functions in mammalian tissues. Hemoglobin scavanges NO and this limits NO's biological activity; conversely, it has recently been proposed that reversible NO binding to the beta- globin cysteine 93 is allosterically linked to oxygenation and this contributes to the control of oxygen delivery in tissues. We have investigated NO transport and metabolism by hemoglobin in human subjects in vitro and in vivo. We have developed new, highly sensitive chemical and spectrophotometric assays for S- nitrosohemoglobin (SNO-Hb) and nitrosyl (heme) hemoglobin (Hb-Fe II-NO). We find that inhalation of NO at 80 ppm by normal subjects leads to formation of significant amounts of nitrosylated hemoglobin with a large arteriovenous gradient, largely due to formation of methemoglobin >> Hb-Fe II-NO SNO- Hb. The primary cause of the A-V difference is the Hb-FE II- NO. In addition, we find an A-V gradient in red cell nitrate and nitrate levels. These results confirm the existence of the SNO-Hb pathway but suggest that reversible binding to ferrous heme is a more important mechanism. Similar results have been obtained with sickle cell patients; in vitro red cell studies with NO show the dominance of methemoglobin formation. We are also using a variety of physiological measures of blood flow- including MRI and reflectance spectroscopy - to examine the effects of NO inhalation on regional blood flow. We have also begun measurements in transgenic/knockout mice manifesting a sickle cell disease-like phenotype. In parallel experiments we have established quantitative mRNA (real-time), protein (Western) and chemiluminescence assays for nitric oxide synthase (NOS) enzymes and their products to identify the tissues in which NO production is occurring in these experimental subjects. We have recently shown that inhaled NO will change blood flow in the arm after NOS synthase inhibition. We expect that these studies will clarify the complex interaction of NO and hemoglobin and establish how hemoglobin participates in the delivery and metabolism of NO. They should also clarify the therapeutic potential of NO in diseases such as sickle cell disease.